Background: Honey bees play a crucial role in pollination and ecological balance. Apis mellifera L. colonies, especially those located in specific geographic regions, such as the palm garden in Eastern Thailand, are susceptible to potential threats from microbial contaminants. Understanding and detecting microbial organisms in these beehives is essential for the preservation of bee health, honey production, and the broader ecosystem. However, the problem of microbial infection and antibiotic-resistant bacteria is more severe and continuously increasing, resulting in a health, economic, and social crisis. The purpose of this study is to determine the prevalence of microorganisms in A. mellifera beehives in palm gardens in Rayong province, Eastern Thailand. Results: Ten swabs in transport media were swabbed and obtained from different parts of each beehive (1 swab per beehive), for a total of 10 hives. Traditional microbial culture-based methods, biochemical tests, and antimicrobial susceptibility (disc-diffusion) tests were used to detect microbial organisms and antibiotic resistance in bacteria. The swab tests from nine beehives resulted in the detection of Gram-positive bacteria (63.64%), Gram-negative bacteria (27.27%), and fungi/yeast (9.09%). These microorganisms are classified as a group of coagulase-negative Staphylococcus spp. and made up 40.91% of the bacteria discovered. Other bacteria found were Coryneform bacteria (13.64%), Pantoea spp. (13.64%), Bacillus spp. (9.09%), yeast (9.09%), glucose non-fermentative Gram-negative bacilli (9.09%), and Pseudomonas spp. (4.55%). However, due to the traditional culture-based and 0biochemical tests usually used to identify the microbial organisms in clinical specimens and the limitation of identifying some environmental microbial species, the results of the antimicrobial susceptibility test cannot reveal if the organism is resistant or susceptible to the drug. Nevertheless, drug-sensitive inhibition zones were formed with each antibiotic agent. Conclusions: Overall, the study supports prevention, healthcare, and public health systems. The contamination of microorganisms in the beehives may affect the quality of honey and other bee products or even the health of the beekeeper. To avoid this kind of contamination, it is therefore necessary to wear personal protective equipment while harvesting honey and other bee products.